Green transgenic fluorescent ornamental fish

ABSTRACT

The present invention relates to transgenic green ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

The present application claims the priority benefit of U.S. provisionalapplication Nos. 61/658,616, filed Jun. 12, 2012, and 61/661,641, filedJun. 19, 2012, each of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to transgenic fish, particularly green transgenicfish.

2. Description of Related Art

Transgenic technology involves the transfer of a foreign gene into ahost organism enabling the host to acquire a new and inheritable trait.Transgenic technology has many potential applications. For example, itcan be used to introduce a transgene into a fish in order to create newvarieties of fish. There are many ways of introducing a foreign geneinto fish, including: microinjection (e.g., Zhu et al., 1985; Du et al.,1992), electroporation (Powers et al., 1992), sperm-mediated genetransfer (Khoo et al., 1992; Sin et al., 1993), gene bombardment or genegun (Zelenin et al., 1991), liposome-mediated gene transfer (Szelei etal., 1994), and the direct injection of DNA into muscle tissue (Xu etal., 1999). The first transgenic fish report was published by Zhu etal., (1985) using a chimeric gene construct consisting of a mousemetallothionein gene promoter and a human growth hormone gene. Most ofthe early transgenic fish studies have concentrated on growth hormonegene transfer with an aim of generating fast growing fish. While amajority of early attempts used heterologous growth hormone genes andpromoters and failed to produce these fish (e.g. Chourrout et al., 1986;Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhancedgrowth of transgenic fish has been demonstrated in several fish speciesincluding Atlantic salmon, several species of Pacific salmons, and loach(e.g. Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).

The black skirt tetra (Gymnocorymbus ternetzi) has been commerciallycultured in the United States at least as early as 1950 (Innes, 1950).However, for the ornamental fish industry the dark striped pigmentationof the adult black skirt tetra does not aid in the efficient display ofthe various colors. The albino black skirt tetra, or “white tetra” is avariant that arose during domestication and shows decreasedpigmentation. The availability of such fish having modified pigmentationfor transgenesis with fluorescent proteins would result in betterproducts for the ornamental fish industry due to better visualization ofthe various colors.

Many fluorescent proteins are known in the art and have been used toinvestigate various cellular processes, including fluorescent proteinsexhibiting various green, red, yellow, blue, or purple colors. Althoughtransgenic experiments involving fluorescent proteins have provided newmarkers and reporters for transgenesis, progress in the field ofdeveloping and producing ornamental fish that express such proteins hasbeen limited.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention concerns making transgenicfluorescent fish and providing such fish to the ornamental fishindustry.

In some embodiments, transgenic fish or methods of making transgenicfish are provided. In certain aspects, the transgenic fish are fertile,transgenic, fluorescent fish. In a particular embodiment, the fish foruse with the disclosed constructs and methods is the white tetra. Tetraskin color is determined by pigment cells in their skin, which containpigment granules called melanosomes (black or brown color), xanthosomes(yellow color), erythrosomes (orange or red color), or iridosomes(iridescent colors, including white color). The number, size, anddensity of the pigment granules per pigment cell influence the color ofthe fish skin. White tetra have diminished number, size, and density ofmelanosomes and hence have lighter skin when compared to the wild typeblack skirt tetra.

In certain specific embodiments there are provided transgenic tetra orprogeny thereof comprising specific transgenic integration events,referred to herein as transformation events. These fish are ofparticular interest because, for example, they embody an aestheticallypleasing green color. Transgenic fish comprising these specifictransgenic events may be homozygous or heterozygous (including, forexample, hemizygous) for the transformation event. Homozygous fish bredwith fish lacking a transformation event will in nearly all casesproduce 100% heterozygous offspring. Eggs, sperm, and embryos comprisingthese specific transgenic events are also included as part of theinvention.

In one such embodiment regarding a specific transgenic integrationevent, a green transgenic tetra or progeny thereof is providedcomprising chromosomally integrated transgenes, wherein the tetracomprises the “Green tetra 1 transformation event,” sperm comprising theGreen tetra 1 transformation event having been deposited as ECACCaccession no. 12061301. The chromosomally integrated transgenes may bepresent on one integrated expression cassette or two or more integratedexpression cassettes. In certain aspects, such a transgenic tetra is afertile, transgenic tetra. In more specific aspects, such a tetra is atransgenic White tetra. Such a transgenic tetra may be homozygous orheterozygous (including, for example, hemizygous) for the transgenes orintegrated expression cassette(s).

Also disclosed are methods of providing a transgenic tetra comprisingthe Green tetra 1 transformation event to the ornamental fish market. Insome embodiments, the method comprises obtaining a transgenic tetra orprogeny thereof comprising chromosomally integrated transgenes, whereinthe tetra comprises the “Green tetra 1 transformation event,” spermcomprising the Green tetra 1 transformation event having been depositedas ECACC accession no. 12061301, and distributing the fish to theornamental fish market. Such fish may be distributed by a grower to acommercial distributor, or such fish may be distributed by a grower or acommercial distributor to a retailer such as, for example, amulti-product retailer having an ornamental fish department.

In some aspects, methods of producing a transgenic tetra are providedcomprising: (a) obtaining a tetra that exhibits fluorescence andcomprises one or more chromosomally integrated transgenes or expressioncassettes, wherein the tetra comprises the “Green tetra 1 transformationevent,” sperm comprising the Green tetra 1 transformation event havingbeen deposited as ECACC accession no. 12061301; and (b) breeding theobtained tetra with a second tetra to provide a transgenic tetracomprising the Green tetra 1 transformation event. The second tetra maybe a transgenic or non-transgenic tetra.

In further embodiments, also provided are methods of producing atransgenic organism, the method comprising using sperm comprising theGreen tetra 1 transformation, such sperm having been deposited as ECACCaccession no. 12061301, to produce transgenic offspring. Such offspringmay be, for example, a tetra, a species of the Gymnocorymbus genus, afish species or genus related to tetra, or another fish species orgenus. In some aspects, the fish may be produced using in vitrofertilization techniques known in the art or described herein.

As used in this specification, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Any embodiment of any of the present methods, kits, and compositions mayconsist of or consist essentially of—rather thancomprise/include/contain/have—the described features and/or steps. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION Transgenic Fish

In some aspects, the invention regards transgenic fish. Methods ofmaking transgenic fish are described in, for example, U.S. Pat. Nos.7,135,613; 7,700,825; 7,834,239, each of which is incorporated byreference in its entirety.

It is preferred that fish belonging to species and varieties of fish ofcommercial value, particularly commercial value within the ornamentalfish industry, be used. Such fish include but are not limited tocatfish, zebrafish and other danios, medaka, carp, tilapia, goldfish,tetras, barbs, sharks (family Cyprinidae), angelfish, loach, koi,glassfish, catfish, discus, eel, tetra, goby, gourami, guppy,Xiphophorus, hatchet fish, Molly fish, or pangasius. A particular fishfor use in the context of the invention is a tetra, Gymnocorymbusternetzi. Tetra are increasingly popular ornamental animals and would beof added commercial value in various colors. Tetra embryos are easilyaccessible and nearly transparent. A fish that is of particular use withthe disclosed constructs and methods is the White Tetra. Tetra skincolor is determined by pigment cells in the skin, which contain pigmentgranules called melanosomes. The number, size, and density of themelanosomes per pigment cell influence the color of the fish skin. WhiteTetra have diminished number, size, and density of melanosomes and hencehave lighter skin when compared to the wild type tetra.

Fertilization from Frozen Sperm

Fish sperm freezing methods are well-known in the art; see, e.g., Walkerand Streisinger (1983) and Draper and Moens (2007), both of which areincorporated herein by reference in their entireties. To obtain thetransgenic fish disclosed herein, frozen tetra sperm may be used tofertilize eggs.

Briefly, one or two breeding pairs of tetra should be placed in ashoebox with an artificial spawning mat. The water level in the shoeboxshould be ˜2-3 inches and kept at 75-85° F. Low salinity (conductivity100-200 uS/cm) and slight acidity (˜pH 6.9) promote spawning. The fishmay be exposed to a natural or artificial light cycle; the photoperiodstarts at 8 am and ends at 10 pm. The following morning, remove anddiscard the eggs. Tetra may be anesthetized by immersion in tricainesolution at 16 mg/100 mL water. After gill movement has slowed, removeone female, rinse it in water, and gently blot the belly damp-dry with apaper towel. The eggs should not be exposed to water as this willprevent fertilization. Gently squeeze out the eggs onto a slightlyconcave surface by applying light pressure to the sides of the abdomenwith a thumb and index finger and sliding the fingers to the genitalpore. Ready to spawn females will release the eggs extremely easily, andcare should be taken not to squeeze the eggs out while blotting thefish. Good eggs are yellowish and translucent; eggs that have remainedin the female too long appear white and opaque. The females will releasethe eggs only for an hour or so. Eggs from several females may bepooled; the eggs can be kept unfertilized for several minutes. The spermis thawed at 33° C. in a water bath for 18-20 seconds. 70 μl roomtemperature Hanks solution is added to the vial and mixed. The sperm isthen immediately added to the eggs and gently mixed. The sperm and eggsare activated by adding 750 μl of fish water and mixing. The mixture isincubated for 5 minutes at room temperature. The dish is then filledwith fish water and incubated at 28° C. After 2-3 hours, fertile embryosare transferred to small dishes where they are further cultured.

Parichy and Johnson, 2001, which is incorporated by reference in itsentirety, provides additional examples regarding in vitro fertilization.

The invention further encompasses progeny of a transgenic fishcontaining the Green tetra 1 transformation event, as well as suchtransgenic fish derived from a transgenic fish egg, sperm cell, embryo,or other cell containing a genomically integrated transgenic construct.“Progeny,” as the term is used herein, can result from breeding twotransgenic fish of the invention, or from breeding a first transgenicfish of the invention to a second fish that is not a transgenic fish ofthe invention. In the latter case, the second fish can, for example, bea wild-type fish, a specialized strain of fish, a mutant fish, oranother transgenic fish. The hybrid progeny of these matings have thebenefits of the transgene for fluorescence combined with the benefitsderived from these other lineages.

The simplest way to identify fish containing the Green tetra 1transformation event is by visual inspection, as the fish in questionwould be green colored and immediately distinguishable fromnon-transgenic fish.

EXAMPLES

Certain embodiments of the invention are further described withreference to the following examples. These examples are intended to bemerely illustrative of the invention and are not intended to limit orrestrict the scope of the present invention in any way and should not beconstrued as providing conditions, parameters, reagents, or startingmaterials that must be utilized exclusively in order to practice the artof the present invention.

Example 1 Green Transgenic Tetra

Transgenic fish exhibiting a green color are provided. The specifictransgenic events embodied in these fish are designated Green tetra 1.Sperm from these fish may be used to fertilize tetra eggs and therebybreed transgenic tetra that comprise these specific transgenicintegration events. Sperm from this line is being deposited at theEuropean Collection of Cell Cultures (ECACC), Porton Down, Salisbury,SP4 OJG, United Kingdom, under the provisions of the Budapest Treaty as“Green tetra 1” (accession no. 12061301).

The fluorescent transgenic fish have use as ornamental fish in themarket. Stably expressing transgenic lines can be developed by breedinga transgenic individual with a wild-type fish, mutant fish, or anothertransgenic fish. The desired transgenic fish can be distinguished fromnon-transgenic fish by observing the fish in white light, sunlight,ultraviolet light, blue light, or any other useful lighting conditionthat allows visualization of the green color of the transgenic fish.

The fluorescent transgenic fish should also be valuable in the marketfor scientific research tools because they can be used for embryonicstudies such as tracing cell lineage and cell migration. Additionally,these fish can be used to mark cells in genetic mosaic experiments andin fish cancer models.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 7,135,613-   U.S. Pat. No. 7,700,825-   U.S. Pat. No. 7,834,239-   Brem et al., Aquaculture, 68:209-219, 1988.-   Chourrout et al., Aquaculture, 51:143-150, 1986.-   Delvin et al., Nature, 371:209-210, 1994.-   Draper and Moens, In: The Zebrafish Book, 5^(th) Ed.; Eugene,    University of Oregon Press, 2007.-   Du et al., Bio/Technology, 10:176-181, 1992.-   Innes, W. T., Exotic Aquarium Fishes: A work of general reference,    Innes Publishing Company, Philadelphia, 1950.-   Gross et al., Aquaculture, 103:253-273, 1992.-   Khoo et al., Aquaculture, 107:1-19, 1992.-   Lamason et al., Science, 310 (5755):1782-1786, 2005.-   Penman et al., Aquaculture, 85:35-50, 1990.-   Powers et al., Mol. Marine Biol. Biotechnol., 1:301-308, 1992.-   Sin et al., Aquaculture, 117:57-69, 1993.-   Szelei et al., Transgenic Res., 3:116-119, 1994.-   Tsai et al., Can. J. Fish Aquat. Sci., 52:776-787, 1995.-   Walker and Streisinger, Genetics 103: 125-136, 1983.-   Xu et al., DNA Cell Biol., 18, 85-95, 1999.-   Zelenin et al., FEBS Lett., 287 (1-2):118-120, 1991.-   Zhu et al., Z. Angew. Ichthyol., 1:31-34, 1985.

What is claimed is:
 1. A transgenic tetra comprising a chromosomallyintegrated expression cassette encoding a green fluorescent protein,wherein the tetra comprises the “Green tetra 1 transformation event,”sperm comprising the Green tetra 1 transformation event having beendeposited as ECACC accession no.
 12061301. 2. The transgenic tetra ofclaim 1, further defined as a fertile, transgenic tetra.
 3. Thetransgenic tetra of claim 1, further defined as a transgenic WhiteTetra.
 4. The transgenic tetra of claim 1, wherein the fish ishomozygous for the integrated expression cassette.
 5. The transgenictetra of claim 1, wherein the fish is heterozygous for the integratedexpression cassette.
 6. A method of providing a transgenic tetra to theornamental fish market, comprising obtaining a transgenic tetra inaccordance with claim 1, and distributing the fish to the ornamentalfish market.
 7. The method of claim 6, wherein the fish are distributedby a grower to a commercial distributor.
 8. The method of claim 6,wherein the fish are distributed by a grower or a commercial distributorto a retailer.
 9. The method of claim 8, wherein the retailer is amulti-product retailer having an ornamental fish department.
 10. Amethod of producing a transgenic tetra comprising: (a) obtaining a tetrathat comprises a chromosomally integrated expression cassette encoding agreen fluorescent protein, wherein the tetra comprises the “Green tetra1 transformation event,” sperm comprising the Green tetra 1transformation event having been deposited as ECACC accession no.12061301; and (b) breeding the obtained tetra with a second tetra toprovide a transgenic tetra comprising the Green tetra 1 transformationevent.
 11. The method of claim 10, wherein the second tetra is anon-transgenic tetra.
 12. A progeny of a transgenic tetra of claim 1that comprises a chromosomally integrated expression cassette encoding agreen fluorescent protein, wherein the tetra and progeny comprises the“Green tetra 1 transformation event,” sperm comprising the Green tetra 1transformation event having been deposited as ECACC accession no.12061301.
 13. The progeny fish of claim 12, further defined as afertile, transgenic tetra.
 14. The progeny fish of claim 12, furtherdefined as a transgenic White Tetra.
 15. The progeny fish of claim 12,wherein the fish is homozygous for the integrated expression cassette.16. The progeny fish of claim 12, wherein the fish is heterozygous forthe integrated expression cassette.
 17. A method of providing atransgenic fish to the ornamental fish market, comprising obtaining aprogeny fish in accordance with claim 12, and distributing the fish tothe ornamental fish market.
 18. The method of claim 17, wherein the fishare distributed by a grower to a commercial distributor.
 19. The methodof claim 17, wherein the fish are distributed by a grower or acommercial distributor to a retailer.
 20. The method of claim 19,wherein the retailer is a multi-product retailer having an ornamentalfish department.
 21. A method of producing a transgenic fish comprising:(a) obtaining a transgenic fish in accordance with claim 12; and (b)breeding the obtained fish with a second fish to provide a transgenicfish comprising the Green tetra 1 transformation event transformationevent.
 22. The method of claim 21, wherein the second fish is anon-transgenic fish.